Planting Adaptability of Brassica napobrassica cv. Huaxi Under Economic Fruit Forest

Qiong MOU Chengjiang PEI Jiahai WU Yousong WU Juan LI Kongzhen LENG Weinan RAN

Abstract［Objectives］ This study was conducted to optimize the extraction process of Sipunculus nudus polysaccharide and study the antioxidant activity of S. nudus polysaccharide.

［Methods］ S. nudus polysaccharide was extracted by ultrasonic microwave assisted method, during which univariate factors including solidliquid ratio, time, temperature, ultrasonic power, microwave power were investigated, to select optimal extracting conditions, which were also verified by an response surface optimization experiment (an fourfactor threelevel test); and the antioxidant activity of S. nudus polysaccharide was studied by determining its DPPH radical scavenging capacity.

［Results］ Through univariate factor investigation and response surface optimization test, it was found that the response surface results were consistent with univariate results. The optimized conditions were as follows: solidliquid ratio at 1∶15, ultrasonic temperature at 60 ℃, ultrasonic power of 360 W and microwave power of 200 W, and the predicted polysaccharide extracting amount was 4.35 mg/g. Under the optimized extracting conditions, the average extracting amount of S. nudus polysaccharide was 4.38 mg/g, which is close to the theoretical predicted value (4.35 mg/g). And the results of this study also showed that different concentrations of polysaccharide solutions have different scavenging effects on DPPH free radical. At the polysaccharide concentration of 0.16 mg/ml, the maximum value of DPPH radical scavenging rate was 7.22%, indicating that polysaccharide has certain antioxidant capacity.

［Conclusions］ This method is a new trial with satisfactory results.

Key wordsUltrasonic microwave; Sipunculus nudus; Polysaccharide; Antioxidant activity



Received: May 18, 2018Accepted: September 27, 2018

Supported by National Natural Science Foundation of Hainan Province (217157); 2017 Provincial College Students Innovation and Entrepreneurship Training Program (201711100005); 2018 Provincial Key Discipline (Thalassochemistry) Construction Fund.

Wen CHEN (1980-), male, P. R. China, associate professor, master, devoted to research about analysis, purification and identification of chemical components in natural products.

*Corresponding author. Email: qilong_chen@163.com.

Sipunculus nudus, also known as Shachong, is a kind of invertebrate wormlike marine coelomate, which is widely distributed in Chinas coastal areas, abundant in Hainan and Guangxi sea areas［1-4］. S. nudus is rich in active substances such as proteins and polysaccharides, and is honored as "the Cordyceps sinensis in the ocean"［5-6］. Polysaccharides are naturally occurring macromolecular compounds constituted by various monosaccharides, widely existing in animals, plants, fungi and microorganisms［7］. Polysaccharides have a variety of biological activity, including antioxidant and antiaging activity, and are capable of protecting some cardiovascular and cerebrovascular systems, improving learning and memory ability, delaying aging, resisting high temperature, promoting lactation, and resisting sepsis［8-10］. They have important applications in biological, food and medical fields. Liu et al.［11］ extracted S. nudus polysaccharide by response surface optimization and ultrasoundassisted extraction, and the results showed that the polysaccharide had antioxidant activity and strong capacity of scavenging DPPH and hydroxyl radicals. Zhang et al.［12］ studied two kinds of polysaccharides, SBP and SWP by infrared spectroscopy, and the two polysaccharides were found to be similar in structure and contain glucose, galactose, xylose and arabinose.

In this study, S. nudus polysaccharide was extracted by ultrasonic microwave assisted method, during which univariate factors including solidliquid ratio, time, temperature, ultrasonic power and microwave power were investigated, to select optimal extracting conditions, which were also verified by an response surface optimization experiment (an fourfactor threelevel test); and the antioxidant activity of S. nudus polysaccharide was studied by determining its DPPH radical scavenging capacity. This study will provide certain theoretical reference for subsequent study of S. nudus polysaccharide.

Materials and Methods

Materials and instruments

Fresh and alive S. nudus (with a length of 4-8 cm and a weight of 3-6 g/individual), the first market of Sanya City; D(+)-anhydrous glucose (AR), Beijing solarbio Science & Technology Co., Ltd.; phenol (AR), Shandong Xiya Chemical Industry Co., Ltd.; concentrated sulfuric acid (AR), Tianjin Hedong Hongyan Reagent Factory; trichloromethane (AR), Xilong Chemical Co., Ltd.; butanol (AR), Shandong Xiya Chemical Industry Co., Ltd.; ethanol (AR), Shandong Xiya Chemical Industry Co., Ltd.; anhydrous ethanol (AR), Guangdong Guanghua SciTech Co., Ltd.; DPPH (BR), Hefei Bomei Biotechnology Co., Ltd; ultrapure water.

Ultrasonicmicrowave chemical reactor XOSM200, Nanjing Xianou Instruments Manufacture Co., Ltd.；thermostat water bath HH, Jiangsu Shenglan Instrument Manufacturing Co., Ltd.; JJ2 triturating machine, Jintan Baita Xinbao Instrument Company; electronic balance FA2204B, Techcomp Precision Balance Instrument Co., Ltd.; ultraviolet and visible spectrophotometer 721G, INESA Analytical Instrument Co., Ltd.; high speed tabletop centrifuge TGL15B, Shanghai Anting Scientific Instrument Factory; circulating water multiuse vacuum pump SHB111, Zhengzhou Greatwall Scientific Industry and Trade Co., Ltd.; rotatory evaporator RE32, Shanghai Yarong Biochemical Instrument Factory; electrothermostatic blast oven DHG9245A, Jiangsu Shenglan Instrument Manufacturing Co., Ltd.; ultravioletvisible spectrophotometer UV2550, Shimadzu (China) Co., Ltd.

Experimental methods

Pretreatment of S. nudus

S. nudus was collected. The tail part of the collected S. nudus was cut off with scissors, and each one was turned over from head to tail with disposable chopsticks, and cleaned for 3-5 times with water to remove sands and then with distilled water for 2-3 times. S. nudus was cut to short sections and prepared at a solidliquid ratio of 1∶1 g/ml, into tissue homogenate, which was preserved at low temperature.

Detection of crude S. nudus polysaccharide

Determination of maximum absorption wavelength of glucose

A certain amount of glucose (100 mg) was accurately weighed, added into a 100 ml volumetric flask and prepared into a glucose reference solution. From the glucose standard solution, 10 ml was pipetted into another 100 ml volumetric flask and diluted to a glucose working solution. Then, 0.00 and 0.60 ml of the glucose working solution were accurately pipetted, respectively, to 50 ml colorimetric tubes, each of which was then added with 2 ml of water, respectively, followed by the addition of 5 ml of concentrated sulfuric acid and 1 ml of phenol solution. The solutions were shaken to mix well and heated in a water bath at 40 ℃ for 15 min. After cooling to room temperature, the maximum absorption wavelength of glucose was determined with UV2550 ultravioletvisible spectrophotometer, with reagent blank as reference.

Preparation of glucose standard curve

0.00, 0.20, 0.40, 0.60, 0.80 and 1.00 ml of the glucose working solution were accurately taken and determined for absorbance at 486 nm by phenolsulfuric acid method, respectively, with reagent blank as reference. A standard curve was plotted, obtaining a regression equation, through which polysaccharide content was calculated.

Determination of S. nudus polysaccharide sample

A certain amount of treated S. nudus sample (5 g) was weighed and extracted by ultrasonic microwave assisted method. The extract was vacuum filtered, obtaining a supernatant, which was concentrated to 1/3 of its original volume and subjected to removal of proteins by Sevag method. The concentrate was added with chloroform and butanol with a ratio at 4∶1, obtaining a mixture, which was centrifuged at 5 000 r/min for 1 min, and the water layer was obtained. The former step was repeated for 3 times. The water layer was added with anhydrous ethanol and stood overnight at 4 ℃. The mixture was then centrifuged at 9 000 r/min for 15 min, obtaining a precipitate, which was dried, obtaining crude S. nudus polysaccharide.

Single factor investigation

Selection of extraction temperature

A certain amount of S. nudus homogenate (5 g) was weighed with an electronic balance into five beakers, respectively. 100 ml of distilled water was added into each beaker, the homogenate in which was then extracted under a microwave power of 150 W and an ultrasonic power of 300 W for 30 min at 40, 50, 60, 70, 80 and 90℃, respectively. Finally, absorbance was determined by phenolsulfuric acid method, followed by the calculation of polysaccharide extracting amount.

Selection of solidliquid ratio

A certain amount of S. nudus homogenate (5 g) was weighed with an electronic balance into five beakers, respectively. The homogenate in each beaker was then extracted under a microwave power of 150 W and an ultrasonic power of 300 W at 60 ℃ at solidliquid ratios of 1∶10, 1∶15, 1∶20, 1∶25 and 1∶30 (g∶ml), respectively. Finally, absorbance was determined by phenolsulfuric acid method, followed by the calculation of polysaccharide extracting amount.

Selection of ultrasonic time

A certain amount of S. nudus homogenate (5 g) was weighed with an electronic balance into five beakers, respectively. 100 ml of distilled water was added into each beaker, the homogenate in which was then extracted under a microwave power of 150 W and an ultrasonic power of 300 W at 60 ℃ for 10, 15, 20, 25 and 30 min, respectively. Finally, absorbance was determined by phenolsulfuric acid method, followed by the calculation of polysaccharide extracting amount.

Selection of ultrasonic power

A certain amount of S. nudus homogenate (5 g) was weighed with an electronic balance into five beakers, respectively. 100 ml of distilled water was added into each beaker, the homogenate in which was then extracted at 60 ℃ for 30 min under a microwave power of 150 W and ultrasonic powers of 240, 300, 360, 420 and 480 W, respectively. Finally, absorbance was determined by phenolsulfuric acid method, followed by the calculation of polysaccharide extracting amount.

Selection of microwave power

A certain amount of S. nudus homogenate (5 g) was weighed with an electronic balance into five beakers, respectively. 100 ml of distilled water was added into each beaker, the homogenate in which was then extracted at 60 ℃ for 30 min under an ultrasonic power of 300 W and microwave powers of 50, 100, 150, 200, 250 and 300 W, respectively. Finally, absorbance was determined by phenolsulfuric acid method, followed by the calculation of polysaccharide extracting amount.

Response surface optimization of extraction conditions

On the basis of above univariate tests, extraction factors having the highest effects on the extracting amount of S. nudus polysaccharide were optimized by a fourfactor threelevel orthogonal test according to the optimal conditions obtained from these univariate tests, and the results were analyzed by response surface and variance analysis.

BoxBehnken design

On the basis of above univariate tests, according to CCD experiment design principle［16］, a fourfactor threelevel orthogonal test was designed using response software DesignExpert.8.0.6.1 with solidliquid ratio (A), ultrasonic temperature (B), ultrasonic power (C) and microwave power (D) as the four factors and the extracting amount of polysaccharide as the response value. The test design and results are shown in Table 1.

Table 1The factors and levels of response surface experiment

Levels

Factors

A Material liquidratio∥g/ml B Temperature℃C Ultrasonicpower∥ WD Microwavepower∥W

-11∶1050300150

01∶1560360200

11∶2070420250

Study on antioxidant activity

Preparation of DPPH and scanning of maximum absorption peak

A certain amount of DPPH reagent (4 mg) was weighed into a 100 ml of volumetric flask and added with anhydrous ethanol to constant volume. The solution was shaken uniformly and cryopreserved in a refrigerator. The solution was scanned with a UV2550 ultravioletvisible spectrophotometer for its maximum absorption wavelength, with anhydrous ethanol as reference.

Effect of S. nudus polysaccharide on DPPHscavenging rate

A certain amount of dried polysaccharide (20 mg) was added with water to constant volume in 50 ml volumetric flasks, respectively. The polysaccharide solution was then diluted to 0.04, 0.08, 0.12, 0.16 and 0.20 mg/ml tobedetected solutions. 2 ml of each of the tobedetected solutions and 3.8 ml of DPPH solution were pipetted into a 50 ml colorimetric tube, and determined at the maximum wavelength for absorbance As after 1 h of reaction at room temperature in the dark; the absorbance of the DPPH solution in 2 ml of distilled water was determined as Ao; and the mixture of 2 ml of each of the tobedetected solutions and 3.8 ml of anhydrous ethanol was determined for absorbance Ar, and added with 1 ml of 5% phenol and 5 ml of concentrated sulfuric acid to allow development.

The scavenging rate of sample to DPPH (DSA) was calculated according to following formula［17］: DSA(%)=［1-(As-Ar)/Ao］×100%.

Results and Analysis

Plotting of glucose standard curve

Determination of maximum absorption wavelength of glucose

The maximum absorption wavelength of glucose standard was determined in the range of 300-700 nm to be 486 nm, which is closer to 490 nm in literature, as shown in Fig. 1.

Liner relation

It could be seen from Fig. 2 that the regression equation of the glucose standard curve was y=52.366x-0.002 6, R2=0.999 8, indicating a good linear relation.

Effect of extracting temperature on extracting amount of crude polysaccharide

It could be seen from Fig. 3 that the optimal polysaccharide extracting temperature was 60 ℃, and the polysaccharide extracting amount increased with the increase of temperature; and when the temperature was 60 ℃, the extracting amount of polysaccharide reached its maximum value, and then gradually decreased with further increase of temperature.



Fig. 1The maximum absorption peak of glucose standard

Table 2The absorbance of glucose standard solution with different concentrations

c∥mg/ml00.002 50.005 00.007 50.010 00.012 5

Absorbance A00.128 00.253 00.392 00.524 00.651 0



Fig. 2The glucose standard curve



Fig. 3Effect of temperature on extracting amount of S. nudus polysaccharide

Agricultural Biotechnology2019

Effect of solidliquid ratio on extracting amount of crude polysaccharide

It could be seen from Fig. 4 that the optimal soliliquid ratio for polysaccharide was 1∶15. The polysaccharide extracting amount gradually increased with the increase of soliliquid ratio, reached its maximum wave at 1∶15, and then gradually decreased with further increase of solidliquid ratio.

Effect of ultrasonic time on extracting amount of crude polysaccharide

It could be seen from Fig. 5 that the optimal ultrasonic time for polysaccharide extraction was 25 min. When the ultrasonic time was short, the extraction of crude polysaccharide was greatly affected. When the ultrasonic time reached 15 min, the effect of extracting time of crude polysaccharide became little. The extracting amount of crude polysaccharide reached its maximum value at the ultrasonic time of 25 min, and then changed at a rate becoming lower and lower.



Fig. 4Effect of soliliquid ratio on extracting amount of S. nudus polysaccharide



Fig. 5Effect of ultrasonic time on extracting amount of S. nudus polysaccharide

Effect of ultrasonic power on extracting amount of crude polysaccharide

It could be seen from Fig. 6 that the optimal ultrasonic power for polysaccharide extraction was 360 W. The polysaccharide extracting amount gradually increased with the increase of ultrasonic power, reached its maximum value at the ultrasonic power of 360 W, and then gradually decreased at a rate becoming lower and lower.



Fig. 6Effect of ultrasonic power on extracting amount of S. nudus polysaccharide

Effect of microwave power on extracting amount of crude polysaccharide

It could be seen from Fig. 7 that the optimal microwave power for polysaccharide extraction was 200 W, and tmicrowave power had a great effect on polysaccharide extraction with large fluctuations. In the microwave power range from 100 to 250 W, the extracting amount of crude polysaccharide was larger; when the microwave power was 50, 150 and 250 W, the polysaccharide extracting amount remained at a lower value; when the microwave power reached 200 W, the polysaccharide extracting amount reached its maximum value; and with further increase of microwave power, the polysaccharide extracting amount gradually decreased, and eventually tended to be stable.



Fig. 7Effect of ultrasonic power on extracting amount of S. nudus polysaccharide

Optimization of S. nudus polysaccharide extracting process by response surface method

BoxBenhken test design and its results

Fitting of response surface test regression equation

On the basis of single factor test results, the extraction conditions of S. nudus polysaccharide were designed using software DesignExpert 8.05 with the extracting rate of S. nudus polysaccharide as response value, and the results are shown in Table 3.

Table 3The design and results of BoxBenhken response surface analysis test

NumberA Ratio ofmaterial toliquid∥g/mlBTemperature℃CUltrasonicpower∥WDMicrowavepower∥WExtractingamountmg/g

11∶15603001502.37

21∶15603602004.36

31∶10603002002.18

41∶10503602002.52

51∶10603601502.43

61∶15503601502.46

71∶15603602004.35

81∶15503002002.38

91∶15604201502.45

101∶15603002502.51

111∶15503602502.35

121∶15703601502.39

131∶15603602004.37

141∶15504202002.30

151∶15703002002.55

(Continued)

(Table 6)

NumberA Ratio ofmaterial toliquid∥g/mlBTemperature℃CUltrasonicpower∥WDMicrowavepower∥WExtractingamountmg/g

161∶20703602002.39

171∶15604202502.29

181∶15703602502.15

191∶20604202002.61

201∶15704202002.28

211∶20603602502.51

221∶10603602502.58

231∶20603601502.29

241∶15603602004.35

251∶10703602002.55

261∶20503602002.51

271∶20603002002.42

281∶10604202002.62

291∶15603602004.36

With the extracting amount of S. nudus polysaccharide as the evaluation standard, analysis was performed with response surface program of DesignExpert.8.05b, and after regression fitting, the regression equation of predicted extracting amount of S. nudus polysaccharide (Y) to independent variables (A, B, C and D) was as below: Y=+4.35-0.010*A-0.016*B+6.522E-003*C-5.578E-003-5.578E-003*D-0.038*A*B-0.065*A*C+0.017*A*D-0.047*B*C-0.034*B*D-0.094*C*D- 0.89*A2 -0.99*B2-0.98*C2 -1.01*D2. The effects of solidliquid ratio (A), temperature, ultrasonic temperature (B), ultrasonic power (C) and microwave power (D) on extracting amount of S. nudus polysaccharide were analyzed by contour map and threedimensional response surface analysis.

Analysis of response surface optimization

According to analysis on the regression model, a contour map and response surface, which could visually reflect the effects of various effects on extracting rate of S. nudus polysaccharide, were obtained after the analysis on solidliquid ratio (A), temperature, ultrasonic temperature (B), ultrasonic power (C) and microwave power (D) using Design Expert 8.05, as shown in Fig. 8-Fig. 13.



(a) The contour map of effects of solidliquid ratio and ultrasonic temperature on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 8Effects of solidliquid ratio and ultrasonic temperature on extracting amount of S. nudus polysaccharide



(a) The contour map of effects of solidliquid ratio and ultrasonic power on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 9Effects of solidliquid ratio and ultrasonic power on extracting amount of S. nudus polysaccharide

It could be seen from Fig. 8 that the slope of the response surface was steep, and the solidliquid ratio significantly interacted with ultrasonic temperature. When fixing ultrasonic temperature at a constant value, the polysaccharide extracting mount increased at first and then decreased with the increase of solidliquid ratio; and when fixing solidliquid ratio at a constant value, the polysaccharide extracting mount increased at first and decreased then with the increase of ultrasonic temperature.

It could be seen from Fig. 9 that the slope of the response surface was steep, and the interaction between solidliquid ratio and ultrasonic power was significant. When fixing solidliquid ratio at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of ultrasonic power; and when fixing ultrasonic power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of solidliquid ratio.

It could be seen from Fig. 10 that the slope of the response surface was steep, and the interaction between solidliquid ratio and microwave power was significant. When fixing solidliquid ratio at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of microwave power; and when fixing microwave power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of solidliquid ratio.



(a) The contour map of effects of solidliquid ratio and microwave power on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 10Effects of solidliquid ratio and microwave power on extracting amount of S. nudus polysaccharide



(a) The contour map of effects of ultrasonic temperature and ultrasonic power on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 11Effects of ultrasonic temperature and ultrasonic power on extracting amount of S. nudus polysaccharide

It could be seen from Fig. 11 that the slope of the response surface was steep, and ultrasonic temperature interacted significantly with ultrasonic power. When fixing ultrasonic temperature at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of ultrasonic power; and when fixing ultrasonic power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of ultrasonic temperature.

It could be seen from Fig. 12 that the slope of the response surface was steep, and ultrasonic temperature interacted significantly with microwave power. When fixing ultrasonic temperature at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of microwave power; and when fixing microwave power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of ultrasonic temperature.

It could be seen from Fig. 13 that the slope of the response surface was steep, and ultrasonic power interacted significantly with microwave power. When fixing ultrasonic power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of microwave power; and when fixing microwave power at a constant value, the polysaccharide extracting amount increased at first and then decreased with the increase of ultrasonic power.



(a) The contour map of effects of ultrasonic temperature and ultrasonic power on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 12Effects of ultrasonic temperature and microwave power on extracting amount of S. nudus polysaccharide



(a) The contour map of effects of ultrasonic temperature and ultrasonic power on extracting amount of S. nudus polysaccharide; (b) the threedimensional response surface.

Fig. 13Effects of ultrasonic power and microwave power on extracting amount of S. nudus polysaccharide

Variance analysis

The 29 groups of tests were subjected to regression analysis with response surface program of DesignExpert.8.05b, and the results of variance analysis and significance analysis of regression model are shown in Table 4. It could be known from response surface analysis that the optimal extraction conditions for S. nudus polysaccharide were solidliquid ratio at 1∶14.97, ultrasonic temperature at 59.92℃, ultrasonic power of 360.26 W and microwave power of 199.86 W. Considering test operability, the extraction conditions were corrected to solidliquid ratio at 1∶15, ultrasonic temperature at 60 ℃, ultrasonic power of 360 W and microwave power of 200 W, and the predicted polysaccharide extracting amount was 4.35 mg/g. It could be seen from Table 3Table 4 that the effects of the four factors on polysaccharide extracting amount ranked as ultrasonic temperature>solidliquid ratio>ultrasonic power>microwave power, and the model showed P<0.0001, R2=0.983 5>0.800, indicating that the regression equation was established successfully, and could be used for prediction and analysis of S. nudus polysaccharide extracting effect, and the quadratic terms of the regression model, A2, B2, C2 and D2 were all very significant.

Table 4The results of variance analysis and significance analysis of regression model

Variance sourceDeviation square sumDegree of freedomMean squareFPSignificance

Model15.67141.1259.530< 0.000 1Extremely significant

ASolidliquid ratio1.249E-00311.249E-0030.0660.800 3Not significant

BTemperature3.224E-00313.224E-0030.1700.685 0Not significant

CUltrasonic power5.104E-00415.104E-0040.0270.871 5Not significant

DMicrowave power3.733E-00413.733E-0040.0200.889 9Not significant

AB5.908E-00315.908E-0030.3100.583 9Not significant

AC0.01710.0170.9100.355 8Not significant

(Continued)

(Table 4)

Variance sourceDeviation square sumDegree of freedomMean squareFPSignificance

AD1.220E-00311.220E-0030.0650.802 6Not significant

BC8.804E-00318.804E-0030.470.504 9Not significant

BD4.527E-00314.527E-0030.240.631 2Not significant

CD0.03510.0351.870.192 7Not significant

A25.1715.17275.22< 0.000 1Extremely significant

B26.3716.37338.95< 0.000 1Extremely significant

C26.2816.28334.04< 0.000 1Extremely significant

D26.5616.56349.19< 0.000 1Extremely significant

Residual0.26140.019

Lack of Fit0.26100.026246.60< 0.000 1

Pure Error4.262E-00441.066E-004

Cor Total15.9328

R20.9835

C.V.%4.99

Adeq Precision21.141

Note: P<0.02: significant difference; P<0.005: highly significant difference; P<0.001: extremely significant difference.

Verification test

The optimized conditions were adopted to extract S. nudus polysaccharide. Three groups were extracted by the same ultrasonic microwave assisted method, and the dried S. nudus polysaccharide was prepared into solutions, which were determined for three times in parallel at 486 nm with a 721G type visible spectrophotometer, followed by calculation of polysaccharide extracting amount.

Table 5The polysaccharide solution absorbance

Number123Average

Extracting amount∥mg/g4.384.394.364.38

It could be seen from Table 5 that under the same optimal extracting conditions, the average extracting amount of S. nudus polysaccharide was 4.38 mg/g, which is close to the theoretical predicted value (4.35 mg/g).

Antioxidant test

Maximum absorption peak of DPPH free radical

It could be seen from Fig. 14 that the maximum absorption peak of DPPH radical was scanned in the range of 400-700 nm［18］, and it was found that DPPH radical has only one absorption peak in this wavelength range, and the maximum absorption wavelength was 517 nm, which is the same as the value in literatures. And the absorbance at this wavelength was 1.574.



Fig. 14The maximum absorption peak of DPPH free radical

Effect of S. nudus polysaccharide on DPPH radical scavenging rate

It could be seen from Fig. 15 that in polysaccharide concentration range of 0.04-0.12 mg/ml, the DPPH free radical scavenging rate was on the decrease. As the polysaccharide concentration increased to 0.16 mg/ml, the DPPH free radical scavenging rate reached its maximum value of 7.22%. It indicated that polysaccharides have a scavenging effect on DPPH free radical, and different concentrations of polysaccharide solutions have different scavenging effects on DPPH free radical.

Table 6The DPPH radical scavenging rate at different concentrations

Concentration∥mg/ml0.040.080.120.160.20

As0.9650.9881.0100.9791.014

A00.9720.9720.9720.9720.972

Ar0.0320.0410.0590.0770.075

Scavenging rate∥%4.0102.4702.0607.2203.190



Fig. 15Effect of S. nudus polysaccharide on free radical scavenging rate of DPPH

Discussion

In this study, S. nudus polysaccharide was extracted by ultrasonic microwave assisted method, during which univariate factors including solidliquid ratio, time, temperature, ultrasonic power and microwave power were investigated, to select optimal extracting conditions. In order to judge the results of univariate tests, a response surface optimization test (a fourfactor threelevel) test was designed to optimize the extraction conditions, and the DPPH radical scavenging rate of S. nudus polysaccharide was determined, to as to study its antioxidant activity.

The maximum absorption wavelength determined in 400-700 nm was 486 nm, and the correlation coefficient R2 of standard curve determined at this wavelength with a visible spectrophotometer was 0.999 8, indicating that the standard curve had good linear relation and could be used for calculation of polysaccharide content. The optimal extraction conditions obtained from univariate investigation were solidliquid ratio at 1∶15 (g∶ml), ultrasonic temperature at 60 ℃, ultrasonic power of 360 W, microwave power of 200 W, and ultrasonic time of 25 min. A response surface optimization test was obtained to verify the extraction conditions, obtaining following optimized conditions: solidliquid ratio at 1∶15.36 (g∶ml), ultrasonic temperature at 59.78℃, ultrasonic power of 364.16 W and microwave power of 192.39 W. Considering test operability, the extraction conditions were corrected to solidliquid ratio at 1∶15, ultrasonic temperature at 60 ℃, ultrasonic power of 360 W and microwave power of 200 W. Variance analysis showed that experimental conditions significantly affected polysaccharide extracting amount. Under corrected extracting conditions, the average extracting amount of S. nudus polysaccharide was 4.38 mg/g, which is close to the theoretical predicted value (4.35 mg/g). And the results of this study also showed that different concentrations of polysaccharide solutions have different scavenging effects on DPPH free radical. At the polysaccharide concentration of 0.16 mg/ml, the maximum value of DPPH radical scavenging rate was 7.22%, indicating that polysaccharide has certain antioxidant capacity.

Conclusions

On the basis of direct water extraction method, S. nudus polysaccharide was extracted by ultrasonic microwave assisted method, during which univariate factors including solidliquid ratio, time, temperature, ultrasonic power and microwave power were investigated, to select optimal extracting conditions, and a response surface optimization test was carried out to determine optimized univariate factors on the basis of the results from univariate tests. S. nudus polysaccharide was then extracted under the optimized conditions and determined with a 721G spectrophotometer for absorbance, followed by the calculation of polysaccharide content through linear regression equation. The test method was mild and could protect polysaccharide activity. It not only can improve extracting rate, but also can reduce operating cost, and the extracting result is satisfactory. This study provides a certain experimental basis and theoretical basis for development of functional foods from S. nudus polysaccharide and further study on medicinal value of S. nudus polysaccharide, and provides a certain experimental and theoretical basis for the development and utilization of S. nudus polysaccharide products, which is of great significance to the research and development of S. nudus.

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